Field of the Invention
This invention relates to the construction of a recombinant African Swine Fever Virus (ASFV) live attenuated candidate strain vaccine for the highly virulent Georgia 2007 isolate ASFV-G. The vaccine comprises the ASFV-G Δ9GLΔUK modified virus, a recombinant ASFV-G modified by deleting a large portion of the 9GL (B119L) gene and the UK (DP96R) gene.
Description of the Relevant Art
African Swine Fever (ASF) is a contagious viral disease of swine. The causative agent, ASF virus (ASFV), is a large enveloped virus containing a double-stranded DNA genome of approximately 190 kilobase pairs. ASFV shares aspects of genome structure and replication strategy with other large double-stranded DNA viruses, including the Poxviridae, Iridoviridae and Phycodnaviridae (Costard et al. 2009. Phil. Trans. Royal Soc. B 364:2683-2696). ASFV infections in domestic pigs are often fatal and are characterized by fever, hemorrhages, ataxia and severe depression. However, the course of infection varies, ranging from highly lethal to sub-clinical, depending on host characteristics and the particular virus strain (Tulman et al. 2009. Curr. Top. Microbiol. Immunol. 328:43-87).
Currently, the disease is endemic in more than twenty sub-Saharan African countries. In Europe, ASF is still endemic on the island of Sardinia (Italy) and new outbreaks have been declared in the Caucasus region since 2007, affecting Georgia, Armenia, Azerbaijan and Russia. Isolated outbreaks have been recently reported in Ukraine, Belarus, Lithuania, Latvia and Poland, posing the risk of further dissemination into neighbouring countries. The epidemic virus, ASFV Georgia 2007/1, is a highly virulent isolate belonging to the genotype II (Chapman et al. 2011. Emerging Infect. Dis. 17:599-605).
Currently, there is no vaccine available for ASF and disease outbreaks are controlled by animal quarantine and slaughter. Attempts to vaccinate animals using infected cell extracts, supernatants of infected pig peripheral blood leukocytes, purified and inactivated virions, infected glutaraldehyde-fixed macrophages, or detergent-treated infected alveolar macrophages failed to induce protective immunity (Coggins, L. 1974. Prog. Med. Virol. 18:48-63; Forman et al. 1982. Arch. Virol. 74:91-100; Kihm et al. 1987. In: African Swine Fever, Becker, Y. (ed), Martinus Nijhoff, Boston, pp 127-144; Mebus, C. A. 1988. Adv. Virus Res. 35:251-269). Homologous protective immunity does develop in pigs surviving viral infection. Pigs surviving acute infection with moderately virulent or attenuated variants of ASFV develop long-term resistance to homologous, but rarely to heterologous, virus challenge (Hamdy and Dardiri. 1984. Am. J. Vet. Res. 45:711-714; Ruiz-Gonzalvo et al. 1981. In: FAO/CEC Expert Consultation in ASF Research, Wilkinson, P. J. (ed), Rome, pp 206-216). Pigs immunized with live attenuated ASF viruses containing engineered deletions of specific ASFV virulence-associated genes were protected when challenged with homologous parental virus. Specifically, individual deletion of UK (DP69R), 23-NL (DP71L), TK (A240L) or 9GL (B119L) genes from the genomes of pathogenic ASF viruses (Malawi Lil-20/1, Pretoriuskop/96/4, E70 and Georgia 2007) markedly attenuated the virus in swine and the animals immunized with these attenuated viruses were protected against challenge with homologous virus (Moore et al. 1998. J. Virol. 72:10310-10315; Lewis et al. 2000. J. Virol. 74:1275-1285; Zsak et al. 1996. J. Virol. 70:8865-8871; Zsak et al. 1998. J. Virol. 72:1028-1035). These observations constitute the only experimental evidence describing the rational development of an effective live attenuated virus against ASFV.
In particular, deletion of 9GL (B119L) in highly virulent ASFV isolates Malawi Lil-20/1, Pretoriuskop/96/4, and Georgia2007 (Lewis et al., supra; Neilan et al. 2004. Virol. 319:337-342; O'Donnell et al. 2015. J. Virol. 89: 8556-8566) resulted in complete attenuation of these viruses in swine. Administration of Malawi Lil-20/1Δ9GL or Pretoriuskop/96/4 Δ9GL or the E70 ΔUK mutants to pigs via IM injection at a relatively high virus dose did not induce clinical signs, with all animals surviving the infection. Furthermore, IM inoculation of pigs with these viruses induced protection against challenge with virulent parental viruses (Zsak et al. 1998, supra; Lewis et al., supra; O'Donnell et al., supra). These observations constitute the only experimental evidence describing the rational development of an effective live attenuated virus against ASFV.
Since there are not ASFV vaccines currently available, the development of any experimental vaccine that may induce any type of protection against the lethal presentation of the disease is of great interest.